Vol. 18, No. 3 Printed in U.SA.
JOURNAL OF VIROLOGY, June 1976, p. 950-955 Copyright © 1976 American Society for Microbiology
Prophage Induction and Inactivation by UV Light B. J. BARNHART,* S. H. COX, AND J. H. JETT Cellular and Molecular Biology Group, Los Alamos Scientific Laboratory, University of California, Los Alamos, New Mexico 87545
Received for publication 20 June 1975
Analysis of the induction
curves
for
UV
light-irradiated Haemophilus in-
fluenzae lysogens and the distribution of pyrimidine dimers in a repair-deficient lysogen suggests that one dimer per prophage-size segment of the host bacterial chromosome is necessary as a preinduction event. The close correlations obtained prompted a renewed consideration of the possibility that direct prophage induction occurs when one dimer is stabilized within the prophage genome. The host excision-repair system apparently functions to reduce the probability of "stabilizing" within the prophage those dimers that are necessary for induction and inactivation. The presence of the inducible defective prophage in strain Rd depresses the inducibility of prophage HPlcl.
Prophages may be induced by physical and chemical agents that alter DNA structure or replication. If DNA of the lysogenic bacterium is altered, the prophage is directly induced, and if altered DNA is introduced into the lysogen by conjugation or transfection (7) or by transformation (15), the prophage may be indirectly induced. It is currently believed that neither excision-repair nor photoreactivation is necessary for induction but that the recA recombinational-repair system is required (8). Relatively low doses of UV radiation bring about induction of prophages in UV-sensitive bacteria deficient in excision-repair (13, 15). The observation that uvr+ lysogens require higher doses of UV radiation for induction has led to the conclusion that the bacterial excisionrepair system acts to decrease the induction response (13, 15). We report here on the direct induction and inactivation of the prophage HPlcl of Haemophilus influenzae by UV light. The frequencies of induced bacteria are correlated with the frequencies of pyrimidine dimers per prophage-size segment of DNA. Inactivation of the induction response at higher UV doses is also considered. MATERIALS AND METHODS Bacterial strains. H. influenzae strain Rd (1) and two derivatives of Rd isolated in this laboratory (2) were used in these experiments. BC100 is a uvrmutant that harbors an inducible defective prophage like the parental strain Rd, and BC200 is a uvr+ mutant that is less sensitive to UV and X-irradiation due to the lack of a defective prophage harbored by Rd (3). Lysogenic stocks of these bacteria were prepared as previously described (3) using the Haemophilus phage HPlcl (11).
Media, diluents, and buffers. All cultures were grown in brain heart infusion medium (5) supplemented with 1 mM CaCl2 (CaBH medium). Difco Eugonbroth (3%, wt/vol), supplemented with 1 mM CaCl2 (CaEb), was used as diluent for titering colony-forming units (CFUs), induced lysogens, and phage PFUs. Bacteria and phage were titered on brain heart infusion agar (Difco) containing added hemin and nicotinamide adenine dinucleotide (5) and 1 mM CaCl2 (CaBH-agar). UV light irradiation. For irradiation, bacteria were centrifuged and suspended at 1 x 108 CFU/ml, and phage were diluted to approximately 109 PFU/ml in UV buffer consisting of 0.01 M PO4 and 0.15 M NaCl (pH 7.1), supplemented with 0.2 ml Tween 80/liter. More than 90% of the UV light at a wavelength of 254 nm was transmitted through 1 mm of these suspensions in 100-mm glass petri dishes. The sources of UV light were an unshielded 30-W General Electric germicidal lamp (G30T8) that emits mainly at 254 nm with an incident dose rate of 20 ergs/mm2 per s and a 15-W General Electric germicidal lamp (G15T8) fitted with a variable aperture, permitting from 1 to 3 ergs/mm2 per s. Suspensions were subjected to manual reciprocal agitation during exposure. The output of the lamps were determined as previously described (3). Calculation of frequency of induced lysogens. In every experiment, a fraction of the lysogens was induced spontaneously (i.e., with no exposure to the UV light used for irradiation). The level of this spontaneous induction varies from one strain of lysogen to another and also depends on handling of the cells during the experiment. The average zerodose induction frequency was 9% for BC100 and BC200. The lowest zero-dose induction frequency observed was 2% and the highest 25%. Thus, to determine the fraction of lysogens induced by the action of UV light, these spontaneous induction frequencies were treated as a background against
950
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UV INDUCTION AND INACTIVATION OF PROPHAGE
which the induction response was measured. However, for the higher UV doses, an increasing fraction of those spontaneously induced lysogens will be inactivated, and only that fraction of the spontaneously induced lysogens that survive should be subtracted. Accordingly, the fraction of viable lysogens [F(D)] induced at dose D was calculated as follows: F (D) =
IC(D)
-
IC(O)e-DID.
(i)
CFU where IC(D) is the number of infective centers induced by dose D; IC(O)e-DID.. is the number of infective centers induced at zero dose corrected for inactivation; CFU is the titer of CFUs; and Di. is the infective center inactivation constant. Di. was obtained by assuming that the spontaneously induced lysogens were inactivated at the same degree as the UV-induced lysogens at the higher UV doses and is the slope of the descending portion of the raw data curve for induction frequency versus dose. Data for each experiment were analyzed separately according to the equation above, and the data available at each dose point were averaged. The data points in Fig. 1 were obtained in this manner and were plotted with error bars that represent the root mean square deviations from the average. Throughout these experiments, the induction frequencies have been measured by determining the fractions of irradiated lysogens that produce phage plaques. This method gives an accurate measurement of the fraction of irradiated bacteria capable of producing at least one phage. Except at relatively high UV doses (i.e., greater than 50 ergs/mm2 for the uvr- lysogen and greater than 300 ergs/mm2 for the uvr+ lysogens), no detectable delay in cell lysis with release of phage has been noted. Even then, the delay was only one to two cell doubling times, an interlude insufficiently long to allow the lawn of indicator bacteria to overgrow the irradiated lysogens and to give artificially lowered induction frequencies. Isolation of pyrimidine dimers. The thyminecontaining dimer content (CT, TT) of irradiated bacterial DNA was determined as previously described (4) utilizing the paper chromatography technique of Setlow et al. (16). Assay for induced lysogens. After resuspension of bacterial strains lysogenic for phage HPlcl in cold UV buffer and irradiation, the bacteria were diluted into ice-cold CaEb to give 103 to 104 induced lysogens/ml. A 0.1-ml portion was then added to 0.1 ml of H. influenzae strain Rd grown to early logarithmic phase (108/ml) in CaBH medium. The bacteria were immediately plated with 3 ml of halfstrength CaBH-agar over solidified CaBH-agar and incubated at 37 C. Assay for phage yield. Irradiated phage were diluted, added to indicator bacteria, and, after 15 min of incubation at 37 C to permit phage adsorption, plated as described above.
RESULTS Induction of prophage HPlcl as a function of UV dose. The fractions of viable centers in-
951
duced by various doses of UV irradiation are presented in Fig. 1. Induction increased in all three cultures at low doses; however, the frequency ofthe irradiated uvr- lysogen producing plaques reached a maximum at 12 ergs/mm2 and decreased rapidly at higher doses. At low UV doses, the uvr+ lysogens were induced with lesser efficiencies than the uvr- culture, but induction frequencies continued to increase at doses that reduced the frequency of uvr- induction. Induction frequencies for the uvr+ lysogens did not begin to level off until doses of 40 to 50 ergs/mm2. Thus, it appears that dimers triggering induction in this system are subject to repair by excision-repair enzymes in the uvr+ lysogens (15). Correlation of pyrimidine dimer frequencies and prophage induction. The genome of H. influenzae has a thymine content of 31 mol%. A single DNA strand of 4 x 108 daltons (6) would have 4 x 108 (0.31) or 1.2 x 108 daltons of thymidylate residues, which is equal to 4 x 105 thymine bases. A UV dose of 100 ergs/mm2 results in 0.07% dimerization of thymine (4), which is 140 dimers per single strand of DNA (i.e., [4 x 105] [7 x 10-41/2), or 280 dimers per double strand of 8 x 108 daltons. Due to the linearity of thymine dimerization as a function of UV dose over the range used in our experiment (18), we can extrapolate downward and equate a frequency of 28 dimers per bacterial chromosome for a dose of 10 ergs/mm2. The prophage HPlcl is approximately 2.5 x 107 daltons of DNA (J. W. Bendler, Ph.D. thesis, Johns Hopkins Univ., Baltimore, Md., 1968), which is 1/32 of the size of the bacterial chromosome. The dose at which there is an average of one dimer per prophage-size segment of DNA is 11.4 ergs/mm2. The size of H. influenzae DNA has also been reported to be twice that used in our calculations (10). If the larger value is used to calculate the number of dimers per bacterial DNA complement, the number of dimers doubles but the prophage to bacterial DNA ratio becomes 1/64. Thus, the UV dose giving one dimer per prophage-size segment of DNA remains at 11.4 ergs/mm2. The method of analysis of prophage induction data employed here has been used previously by Franklin (9) to describe the induction of a prophage in Escherichia coli. This method assumes that both the induction and inactivation processes are being evoked simultaneously and independently. The inactivation process is described by S(D) = 1 - (1 - e DIDo)n, where S(D) is the fraction of the induced lysogens surviving at dose D, Do is the D37 dose, and n is the extrapolation number. The activation
952
J. VIROL.
BARNHART, COX, AND JETT I.00
a)
010
0.04
0
20
40
60
80
100
120
U V Dose (erg /mm2) FIG. 1. Frequencies of induced lysogenic H. influenzae strains as a function of UV light dose. Two repair-proficient strains, Rd (HPJcl) (A) and BC200 (HP1cl) (a), and one repair-deficient strain, BC100 (HPlcl) (c), were grown to early logarithmic phase, resuspended in UV buffer at titers of approximately 108 CFUs, and irradiated with 254-nm UV light. Aliquots (0.1 ml) were diluted immediately after irradiation into cold CaEb and plated with phage-producing bacteria. The CFU titer was determined just prior to irradiation. All data points, except the two lowest dose BC100 data points, represent averages of up to six separate experiments, and the error bars are standard deviations of the average values.
process is described by I(D) = 1 - e DIDt, where I(D) is the fraction of lysogens induced at dose D, and Da,t is the dose at which 37% of the lysogens are not induced. When both induction and inactivation are occurring simultaneously and independently, the result is described by: F(D) = (1 - e-DIDac,) (1 - [1 - e D/oD]) (ii) where F(D) is the fraction of lysogens induced as determined experimentally (9). This relationship was fit to the data obtained in these experiments by a nonlinear least-squares technique. The resulting parameters are shown in Table 1. This relationship fits the data very well for each of the three sets of data, as indicated by values of the weighted variance obtained that were between 0.7 and 1.6. Thus, the model of simultaneous induction and inactivation describes the data obtained for all three strains. The significance of the values obtained for the parameters and their relationship to dimer formation frequencies is discussed later. Comparison of inactivation of inducible lysogens with inactivation of the phage genome. If the inactivation component of the uvr- induction curve is due to damage to the prophage itself, then a curve of a similar slope might be
TABLE 1. Parameters obtained by fitting equation (ii) to the experimental data points Strain
BC100 BC200 Rd
D,,a,mm(ergs/ 2)
D,, (ergs/mm2)
n
11.3 ± 0.74 51.5 ± 2.8 70.0 ± 5.3
30.4 + 1.18 280 + 20 300 ± 20
1.0 (fixed) 1.9 ± 0.22 1.5 ± 0.20
expected for the inactivation of free phage particles. In Fig. 2 are presented the results of an experiment designed to compare the inactivation of free phage and inducible lysogens. The slopes given as D37 values of the straightline portions are similar for lysogen and phage inactivation, differing only by a factor of 1.33 for the uvr- strain. The inactivation slopes for uvr- and uvr+ lysogens differ by a factor of 7.7, and inactivation of free phage on uvr- and uvr+ hosts gives D37 values differing by more than sixfold. Thus, it seems that our results usingH. influenzae HPlcl lysogens support the conclusion of Mattern et al. (13) for the E. coli-X system that inactivation of lysogens is affected by damage to the prophage. DISCUSSION Induction of Haemophilus phage HPlcl, as measured by assaying the plaque-forming
VOL. 18, 1976
UV INDUCTION AND INACTIVATION OF PROPHAGE
953
4) 0
a. ~0
.-c
10
0.10
0'
C
cn C)
0
c 0
L-IU
.) 0
LL
200
300
400
500
UV Dose (erg /mm2) FIG. 2. Comparison of inactivation of inducible lysogens with inactivation of the phage genome. Cultures of BC100 (HP1ci) uvr- (@) and BC200 (HP1ci) uvr+ (U) were grown, irradiated with UV light, and titered as described in the legend to Fig. 1. Phase inactivation was determined by irradiating 109 HPZci phage per ml in UV buffer and titrating for PFUs on the phage-sensitive strains BC100 uvr- (V) and BC200 uvr+ (A). The numbers in parentheses are the D3, values for the straight-line portions of each curve. The data averages and error bars for the induction curves are described in the legend to Fig. 1. Each data point for free phage survival is the average of two separate experiments.
ability of irradiated lysogenic bacteria, does not require an excision-repair capability of the host. However, the induction response is depressed by the presence of such a repair system (13, 15). The results in Fig. 1 show that the uvr- lysogen BC100 (HPlcl) is induced more efficiently at low UV doses, reaching a maximum induction frequency of approximately 50% at 12 ergs/mm2. At higher doses, the observed induction frequency decreases rapidly, whereas that of the uvr+ lysogens continues to increase.
Analysis of the data obtained for the uvrstrain leads to the conclusion that one dimer per prophage-size piece of DNA triggers induction. In the hit theory of radiation damage, the parameters DQ(,. and Do are the doses at which, on the average, there is one hit per target for induction and inactivation, respectively. For the BC100 strain, the value of Dac, obtained (see Table 1) was 11.3 ergs/mm2. At that dose, there is an average of one hit per target for induction. By assuming that the triggering event is the production of a thymine dimer and using the dose dependence of thymine dimers given above, one is led to the conclusion that
the target size for induction is a prophage-size piece of DNA of 2.5 x 107 daltons. This is to be compared to the Dact value for the uvr+ strain of 51.5 ergs/mm2. Thus, the dose required to induce the uvr+ strain to a given level of induction is 51.5/11.3 = 4.6 times higher than the dose required to induce the uvr- strain to the same level. If the target size is the same in both strains and it is assumed that one dimer is the triggering event, then one is led to the conclusion that approximately four out of five dimers are rendered ineffective or, conversely, that one in five is stabilized so as to trigger the induction response in the uvr+ strain. For the purposes of this discussion, a "stabilized dimer" shall be one which has been formed but not repaired and, if subjected to the action of a repair enyme(s), it will be considered to be in a repair state at some step in excision-repair. Our results (Table 1) also indicate that strain Rd requires a still higher dose to elicit the induction response (Dact = 70 ergs/mm2). This may reflect the induction of the defective prophage in this strain (4). Induction of the defective prophage could result in a lower HPlcl induc-
954
J. VIROL. BARNHART, COX, AND JETV
tion frequency in the population; therefore, the maximum induction frequency for Rd lysogens would be less than that for the defective prophage-free strain BC200. A trend in this direction is suggested in Fig. 1. The 100% induction level is never observed, due to the increased likelihood of inactivation of the induction response at elevated doses. The following consideration raises the possibility that photodimers within the prophage also lead to non-plaque-forming lysogens. In equation (ii), the parameter Do is the D37 dose for inactivation in the straight-line portion of the curve, and n is the extrapolation number that differs from 1.0, producing a shouldered survival curve when repair mechanisms are functioning. When equation (ii) was fit to the BC100 data, n was held constant at 1.0. The value of Do obtained was 30.4 ergs/mm2. Assuming that a single hit produces inactivation would imply that the target size for inactivation is 30.4/11.3 = 2.7 times smaller than the target for induction. This is equivalent to one-third of the prophage genome. The data for both the uvr+ strains are described by approximately the same parameters: Do = 300 ergs/mm2 and n = 2. Thus, there is a factor of 10 decrease in the inactivation sensitivity of these repair-proficient strains when compared to the uvr- strain. Further, the values for n obtained are greater than 1.0, which implies that more than one hit is required. Inactivation could result from the lesion rendering ineffective, by whatever mechanism, any influence of an inducing protein. Inactivation could also result from a stabilized dimer within the prophage (15) interfering with transcription of essential genes or with replication of the prophage genome. Curves describing the UV inactivation of free phage particles, as assayed on uvr- and uvr+ hosts, were compared with those for inactivation of induced lysogens (Fig. 2). The slopes of the uvr- inactivation curves for free phage and lysogens differ by a factor of 1.33. We interpret these results to mean that the target size for rendering the lysogenic bacteria noninducible is approximately the same as the target size for inactivation of free phage particles. As we stated previously, the statistical approach of correlating expected frequencies of HPlcl induction with observed frequencies in a repair-deficient host as a function of UV dose leads us to consider that the occurrence of one dimer within a prophage-size (2.5 x 107 daltons) length of DNA triggers induction. Whether the 2.5 x 107-daltons length is that portion of the bacterial genome occupied by the prophage or is a segment occupied by nonphage
genes is, of course, conjecture. In 1954, Franklin (9) concluded that induction of phage X might involve the activation or inactivation of a particular enzyme-forming system. The appearance of an active inducing protein, as postulated by Tomizawa and Ogawa (17), could satisfy this possibility. Franklin (9) also offered the possibility that induction might result from the direct activation of provirus. Our results suggest that the induction response may indeed result from the appearance of UV photodimer(s) within the prophage genome of 2.5 x 107 daltons. This is the size segment of DNA that our results and correlations show to be "induction sensitive." Although the current dogma does not embrace the notion of prophage induction as being triggered by an initial event ocurring within the prophage genome, we feel that the close correlation of induction frequencies in a repair-deficient lysogen with dimer frequencies per prophage-size piece of the bacterial genome justifies a renewed consideration of induction being the result of a radiation insult primarily and directly to the prophage itself. We can only speculate as to the mechanism of action of the inducing lesion. One dimer within the prophage could render the repressoroperator complex (12) ineffective so that transcription and replication of phage genes proceed. Among the early proteins transcribed would be one that inactivates repressor protein in both the bound and unbound states. However, if induction is mediated by an inducing protein, as suggested by Tomizawa and Ogawa (17), the inducing dimer could permit a receptor on the prophage or on the repressor itself to become available to binding and thereby subject to the functional influence of the inducing protein. This protein could be produced as the result of UV lesions in any stretch of DNA, whereas a lesion specifically within the prophage would be necessary to permit such an inducing protein to function on the repressed operon. Whether induction involves recombinational repair, as described by Rupp and HowardFlanders (14), or an event similar to one of the steps in recombination that acts to lift phage immunity, is not known. Since H. influenzae is rec+ (15), it is possible that a gene product similar to that coded for by recA of E. coli acts on the UV lesion incurred by prophage HPlcl in a manner that permits prophage derepression. ACKNOWLEDGMENTS We thank Richard T. Okinaka of this laboratory and Thomas S. Matney of the M. D. Anderson Hospital and Tumor Institute, University of Texas, Houston, for helpful
VOL. 18, 1976
UV INDUCTION AND INACTIVATION OF PROPHAGE
discussions while this work was in progress and during preparation of the manuscript. This work was performed under the auspices of the U.S. Energy Research and Development Administration. LITERATURE CITED 1. Alexander, H. E., and G. Leidy. 1953. Induction of streptomycin resistance in sensitive Henophilu inluenzae by extracts containing deoxyribonucleic acid
from resistant Henophilus influenzae. J. Exp. Med.
97:17-31. 2. Barnhart, B. J., and S. H. Cox. 1968. Radiation-sensitive
and radiation-resistant mutants of Haemophilus influenzae. J. Bacteriol. 96:280-282.
3. Barnhart, B. J., and S. H. Cox. 1970. Recovery of Haemophilus influenzae from ultraviolet and x-ray damage. Photochem. Photobiol. 11:147-162. 4. Brnhart, B. J., and S. H. Cox. 1970. Radiation sensitivity of Haemophilus influenzae: a composite response. J. Bacteriol. 103:9-15. 5. Barmhart, B. J., and R. M. Heriott. 1963. Penetration of
6. 7. 8.
9.
deoyribonucleic acid into Haemophilus influenzae. Biochim. Biophys. Acta 76:25-39. Bern., H. I., and C. A. Thomas, Jr. 1965. Isolation of high molecular weight DNA from Haemophilus influenzae. J. Mol. Biol. 11:476490. Borek, E., and A. Ryan. 1960. The transfer of a biologically active irradiation product from cell to cell. Biochim. Biophys. Acta 41:67-73. Brooks, K., and A. J. Clark. 1967. Behavior of A bacteriophage in a recombination deficient strain of Esherichia coli. J. Virol. 1:283-293. Franklin, R. 1954. The action spectrum for the ultraviolet
10.
11.
12. 13. 14.
15.
16. 17. 18.
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induction of lysis in Escherichia coli K-12. Biochim. Biophys. Acta 13:137-138. Gillis, M., J. DeLey, and M. DeCleeve. 1970. The determination of molecular weight of bacterial genome DNA from renaturation rates. Eur. J. Biochem. 12: 143-153. Hann, W., and C. S. Rupert. 1963. Infection of transormable cells of Haemophilus influerzae by bacteriophage and bacteriophage DNA. Z. Vererbungpl. 94:336-348. Jacob, F., and J. Monod. 1961. Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3:318-356. Mattern, I. E., M. P. Van Winden, and A. Rorwch. 1965. The range of action of genes controlling radiation sensitivity inEscherichia coli. Mutat. Res. 2:111-131. Rup, D., and P. Howard-Flanders. 1968. Disa6ntinuities in the DNA synthesized in an excision-defective strain of Escherichia col following ultraviolet irradiation. J. Mol. Biol. 31:291-304. Setlow, J. K., M. E. Boling, D. P. Allison, and K. L. Beattie. 1973. Relationship between prophage induction and transfornation in Haemophilus influenzae. J. Bacteriol. 115&153-161. Setlow, R. B., P. A. Swenson, and W. L. Carrier. 1963. Thymine dimers and inhibition of DNA synthesis by ultraviolet irradiation of cells. Science 142:1464-1466. Tonizawa, J. I., and T. Ogawa. 1967. Effect of ultraviolet irradiation on bacteriophage lamnbda immunity. J. Mol. Biol. 23:247-263. Wulff, D. L. 1963. The role of thymine dimer in the photo-inactivation of the bacteriophage T4V,. J. Mol. Biol. 7:431-441.
JOURNAL OF VIROLOGY, June 1976, p. 950-955 Copyright © 1976 American Society for Microbiology
Prophage Induction and Inactivation by UV Light B. J. BARNHART,* S. H. COX, AND J. H. JETT Cellular and Molecular Biology Group, Los Alamos Scientific Laboratory, University of California, Los Alamos, New Mexico 87545
Received for publication 20 June 1975
Analysis of the induction
curves
for
UV
light-irradiated Haemophilus in-
fluenzae lysogens and the distribution of pyrimidine dimers in a repair-deficient lysogen suggests that one dimer per prophage-size segment of the host bacterial chromosome is necessary as a preinduction event. The close correlations obtained prompted a renewed consideration of the possibility that direct prophage induction occurs when one dimer is stabilized within the prophage genome. The host excision-repair system apparently functions to reduce the probability of "stabilizing" within the prophage those dimers that are necessary for induction and inactivation. The presence of the inducible defective prophage in strain Rd depresses the inducibility of prophage HPlcl.
Prophages may be induced by physical and chemical agents that alter DNA structure or replication. If DNA of the lysogenic bacterium is altered, the prophage is directly induced, and if altered DNA is introduced into the lysogen by conjugation or transfection (7) or by transformation (15), the prophage may be indirectly induced. It is currently believed that neither excision-repair nor photoreactivation is necessary for induction but that the recA recombinational-repair system is required (8). Relatively low doses of UV radiation bring about induction of prophages in UV-sensitive bacteria deficient in excision-repair (13, 15). The observation that uvr+ lysogens require higher doses of UV radiation for induction has led to the conclusion that the bacterial excisionrepair system acts to decrease the induction response (13, 15). We report here on the direct induction and inactivation of the prophage HPlcl of Haemophilus influenzae by UV light. The frequencies of induced bacteria are correlated with the frequencies of pyrimidine dimers per prophage-size segment of DNA. Inactivation of the induction response at higher UV doses is also considered. MATERIALS AND METHODS Bacterial strains. H. influenzae strain Rd (1) and two derivatives of Rd isolated in this laboratory (2) were used in these experiments. BC100 is a uvrmutant that harbors an inducible defective prophage like the parental strain Rd, and BC200 is a uvr+ mutant that is less sensitive to UV and X-irradiation due to the lack of a defective prophage harbored by Rd (3). Lysogenic stocks of these bacteria were prepared as previously described (3) using the Haemophilus phage HPlcl (11).
Media, diluents, and buffers. All cultures were grown in brain heart infusion medium (5) supplemented with 1 mM CaCl2 (CaBH medium). Difco Eugonbroth (3%, wt/vol), supplemented with 1 mM CaCl2 (CaEb), was used as diluent for titering colony-forming units (CFUs), induced lysogens, and phage PFUs. Bacteria and phage were titered on brain heart infusion agar (Difco) containing added hemin and nicotinamide adenine dinucleotide (5) and 1 mM CaCl2 (CaBH-agar). UV light irradiation. For irradiation, bacteria were centrifuged and suspended at 1 x 108 CFU/ml, and phage were diluted to approximately 109 PFU/ml in UV buffer consisting of 0.01 M PO4 and 0.15 M NaCl (pH 7.1), supplemented with 0.2 ml Tween 80/liter. More than 90% of the UV light at a wavelength of 254 nm was transmitted through 1 mm of these suspensions in 100-mm glass petri dishes. The sources of UV light were an unshielded 30-W General Electric germicidal lamp (G30T8) that emits mainly at 254 nm with an incident dose rate of 20 ergs/mm2 per s and a 15-W General Electric germicidal lamp (G15T8) fitted with a variable aperture, permitting from 1 to 3 ergs/mm2 per s. Suspensions were subjected to manual reciprocal agitation during exposure. The output of the lamps were determined as previously described (3). Calculation of frequency of induced lysogens. In every experiment, a fraction of the lysogens was induced spontaneously (i.e., with no exposure to the UV light used for irradiation). The level of this spontaneous induction varies from one strain of lysogen to another and also depends on handling of the cells during the experiment. The average zerodose induction frequency was 9% for BC100 and BC200. The lowest zero-dose induction frequency observed was 2% and the highest 25%. Thus, to determine the fraction of lysogens induced by the action of UV light, these spontaneous induction frequencies were treated as a background against
950
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which the induction response was measured. However, for the higher UV doses, an increasing fraction of those spontaneously induced lysogens will be inactivated, and only that fraction of the spontaneously induced lysogens that survive should be subtracted. Accordingly, the fraction of viable lysogens [F(D)] induced at dose D was calculated as follows: F (D) =
IC(D)
-
IC(O)e-DID.
(i)
CFU where IC(D) is the number of infective centers induced by dose D; IC(O)e-DID.. is the number of infective centers induced at zero dose corrected for inactivation; CFU is the titer of CFUs; and Di. is the infective center inactivation constant. Di. was obtained by assuming that the spontaneously induced lysogens were inactivated at the same degree as the UV-induced lysogens at the higher UV doses and is the slope of the descending portion of the raw data curve for induction frequency versus dose. Data for each experiment were analyzed separately according to the equation above, and the data available at each dose point were averaged. The data points in Fig. 1 were obtained in this manner and were plotted with error bars that represent the root mean square deviations from the average. Throughout these experiments, the induction frequencies have been measured by determining the fractions of irradiated lysogens that produce phage plaques. This method gives an accurate measurement of the fraction of irradiated bacteria capable of producing at least one phage. Except at relatively high UV doses (i.e., greater than 50 ergs/mm2 for the uvr- lysogen and greater than 300 ergs/mm2 for the uvr+ lysogens), no detectable delay in cell lysis with release of phage has been noted. Even then, the delay was only one to two cell doubling times, an interlude insufficiently long to allow the lawn of indicator bacteria to overgrow the irradiated lysogens and to give artificially lowered induction frequencies. Isolation of pyrimidine dimers. The thyminecontaining dimer content (CT, TT) of irradiated bacterial DNA was determined as previously described (4) utilizing the paper chromatography technique of Setlow et al. (16). Assay for induced lysogens. After resuspension of bacterial strains lysogenic for phage HPlcl in cold UV buffer and irradiation, the bacteria were diluted into ice-cold CaEb to give 103 to 104 induced lysogens/ml. A 0.1-ml portion was then added to 0.1 ml of H. influenzae strain Rd grown to early logarithmic phase (108/ml) in CaBH medium. The bacteria were immediately plated with 3 ml of halfstrength CaBH-agar over solidified CaBH-agar and incubated at 37 C. Assay for phage yield. Irradiated phage were diluted, added to indicator bacteria, and, after 15 min of incubation at 37 C to permit phage adsorption, plated as described above.
RESULTS Induction of prophage HPlcl as a function of UV dose. The fractions of viable centers in-
951
duced by various doses of UV irradiation are presented in Fig. 1. Induction increased in all three cultures at low doses; however, the frequency ofthe irradiated uvr- lysogen producing plaques reached a maximum at 12 ergs/mm2 and decreased rapidly at higher doses. At low UV doses, the uvr+ lysogens were induced with lesser efficiencies than the uvr- culture, but induction frequencies continued to increase at doses that reduced the frequency of uvr- induction. Induction frequencies for the uvr+ lysogens did not begin to level off until doses of 40 to 50 ergs/mm2. Thus, it appears that dimers triggering induction in this system are subject to repair by excision-repair enzymes in the uvr+ lysogens (15). Correlation of pyrimidine dimer frequencies and prophage induction. The genome of H. influenzae has a thymine content of 31 mol%. A single DNA strand of 4 x 108 daltons (6) would have 4 x 108 (0.31) or 1.2 x 108 daltons of thymidylate residues, which is equal to 4 x 105 thymine bases. A UV dose of 100 ergs/mm2 results in 0.07% dimerization of thymine (4), which is 140 dimers per single strand of DNA (i.e., [4 x 105] [7 x 10-41/2), or 280 dimers per double strand of 8 x 108 daltons. Due to the linearity of thymine dimerization as a function of UV dose over the range used in our experiment (18), we can extrapolate downward and equate a frequency of 28 dimers per bacterial chromosome for a dose of 10 ergs/mm2. The prophage HPlcl is approximately 2.5 x 107 daltons of DNA (J. W. Bendler, Ph.D. thesis, Johns Hopkins Univ., Baltimore, Md., 1968), which is 1/32 of the size of the bacterial chromosome. The dose at which there is an average of one dimer per prophage-size segment of DNA is 11.4 ergs/mm2. The size of H. influenzae DNA has also been reported to be twice that used in our calculations (10). If the larger value is used to calculate the number of dimers per bacterial DNA complement, the number of dimers doubles but the prophage to bacterial DNA ratio becomes 1/64. Thus, the UV dose giving one dimer per prophage-size segment of DNA remains at 11.4 ergs/mm2. The method of analysis of prophage induction data employed here has been used previously by Franklin (9) to describe the induction of a prophage in Escherichia coli. This method assumes that both the induction and inactivation processes are being evoked simultaneously and independently. The inactivation process is described by S(D) = 1 - (1 - e DIDo)n, where S(D) is the fraction of the induced lysogens surviving at dose D, Do is the D37 dose, and n is the extrapolation number. The activation
952
J. VIROL.
BARNHART, COX, AND JETT I.00
a)
010
0.04
0
20
40
60
80
100
120
U V Dose (erg /mm2) FIG. 1. Frequencies of induced lysogenic H. influenzae strains as a function of UV light dose. Two repair-proficient strains, Rd (HPJcl) (A) and BC200 (HP1cl) (a), and one repair-deficient strain, BC100 (HPlcl) (c), were grown to early logarithmic phase, resuspended in UV buffer at titers of approximately 108 CFUs, and irradiated with 254-nm UV light. Aliquots (0.1 ml) were diluted immediately after irradiation into cold CaEb and plated with phage-producing bacteria. The CFU titer was determined just prior to irradiation. All data points, except the two lowest dose BC100 data points, represent averages of up to six separate experiments, and the error bars are standard deviations of the average values.
process is described by I(D) = 1 - e DIDt, where I(D) is the fraction of lysogens induced at dose D, and Da,t is the dose at which 37% of the lysogens are not induced. When both induction and inactivation are occurring simultaneously and independently, the result is described by: F(D) = (1 - e-DIDac,) (1 - [1 - e D/oD]) (ii) where F(D) is the fraction of lysogens induced as determined experimentally (9). This relationship was fit to the data obtained in these experiments by a nonlinear least-squares technique. The resulting parameters are shown in Table 1. This relationship fits the data very well for each of the three sets of data, as indicated by values of the weighted variance obtained that were between 0.7 and 1.6. Thus, the model of simultaneous induction and inactivation describes the data obtained for all three strains. The significance of the values obtained for the parameters and their relationship to dimer formation frequencies is discussed later. Comparison of inactivation of inducible lysogens with inactivation of the phage genome. If the inactivation component of the uvr- induction curve is due to damage to the prophage itself, then a curve of a similar slope might be
TABLE 1. Parameters obtained by fitting equation (ii) to the experimental data points Strain
BC100 BC200 Rd
D,,a,mm(ergs/ 2)
D,, (ergs/mm2)
n
11.3 ± 0.74 51.5 ± 2.8 70.0 ± 5.3
30.4 + 1.18 280 + 20 300 ± 20
1.0 (fixed) 1.9 ± 0.22 1.5 ± 0.20
expected for the inactivation of free phage particles. In Fig. 2 are presented the results of an experiment designed to compare the inactivation of free phage and inducible lysogens. The slopes given as D37 values of the straightline portions are similar for lysogen and phage inactivation, differing only by a factor of 1.33 for the uvr- strain. The inactivation slopes for uvr- and uvr+ lysogens differ by a factor of 7.7, and inactivation of free phage on uvr- and uvr+ hosts gives D37 values differing by more than sixfold. Thus, it seems that our results usingH. influenzae HPlcl lysogens support the conclusion of Mattern et al. (13) for the E. coli-X system that inactivation of lysogens is affected by damage to the prophage. DISCUSSION Induction of Haemophilus phage HPlcl, as measured by assaying the plaque-forming
VOL. 18, 1976
UV INDUCTION AND INACTIVATION OF PROPHAGE
953
4) 0
a. ~0
.-c
10
0.10
0'
C
cn C)
0
c 0
L-IU
.) 0
LL
200
300
400
500
UV Dose (erg /mm2) FIG. 2. Comparison of inactivation of inducible lysogens with inactivation of the phage genome. Cultures of BC100 (HP1ci) uvr- (@) and BC200 (HP1ci) uvr+ (U) were grown, irradiated with UV light, and titered as described in the legend to Fig. 1. Phase inactivation was determined by irradiating 109 HPZci phage per ml in UV buffer and titrating for PFUs on the phage-sensitive strains BC100 uvr- (V) and BC200 uvr+ (A). The numbers in parentheses are the D3, values for the straight-line portions of each curve. The data averages and error bars for the induction curves are described in the legend to Fig. 1. Each data point for free phage survival is the average of two separate experiments.
ability of irradiated lysogenic bacteria, does not require an excision-repair capability of the host. However, the induction response is depressed by the presence of such a repair system (13, 15). The results in Fig. 1 show that the uvr- lysogen BC100 (HPlcl) is induced more efficiently at low UV doses, reaching a maximum induction frequency of approximately 50% at 12 ergs/mm2. At higher doses, the observed induction frequency decreases rapidly, whereas that of the uvr+ lysogens continues to increase.
Analysis of the data obtained for the uvrstrain leads to the conclusion that one dimer per prophage-size piece of DNA triggers induction. In the hit theory of radiation damage, the parameters DQ(,. and Do are the doses at which, on the average, there is one hit per target for induction and inactivation, respectively. For the BC100 strain, the value of Dac, obtained (see Table 1) was 11.3 ergs/mm2. At that dose, there is an average of one hit per target for induction. By assuming that the triggering event is the production of a thymine dimer and using the dose dependence of thymine dimers given above, one is led to the conclusion that
the target size for induction is a prophage-size piece of DNA of 2.5 x 107 daltons. This is to be compared to the Dact value for the uvr+ strain of 51.5 ergs/mm2. Thus, the dose required to induce the uvr+ strain to a given level of induction is 51.5/11.3 = 4.6 times higher than the dose required to induce the uvr- strain to the same level. If the target size is the same in both strains and it is assumed that one dimer is the triggering event, then one is led to the conclusion that approximately four out of five dimers are rendered ineffective or, conversely, that one in five is stabilized so as to trigger the induction response in the uvr+ strain. For the purposes of this discussion, a "stabilized dimer" shall be one which has been formed but not repaired and, if subjected to the action of a repair enyme(s), it will be considered to be in a repair state at some step in excision-repair. Our results (Table 1) also indicate that strain Rd requires a still higher dose to elicit the induction response (Dact = 70 ergs/mm2). This may reflect the induction of the defective prophage in this strain (4). Induction of the defective prophage could result in a lower HPlcl induc-
954
J. VIROL. BARNHART, COX, AND JETV
tion frequency in the population; therefore, the maximum induction frequency for Rd lysogens would be less than that for the defective prophage-free strain BC200. A trend in this direction is suggested in Fig. 1. The 100% induction level is never observed, due to the increased likelihood of inactivation of the induction response at elevated doses. The following consideration raises the possibility that photodimers within the prophage also lead to non-plaque-forming lysogens. In equation (ii), the parameter Do is the D37 dose for inactivation in the straight-line portion of the curve, and n is the extrapolation number that differs from 1.0, producing a shouldered survival curve when repair mechanisms are functioning. When equation (ii) was fit to the BC100 data, n was held constant at 1.0. The value of Do obtained was 30.4 ergs/mm2. Assuming that a single hit produces inactivation would imply that the target size for inactivation is 30.4/11.3 = 2.7 times smaller than the target for induction. This is equivalent to one-third of the prophage genome. The data for both the uvr+ strains are described by approximately the same parameters: Do = 300 ergs/mm2 and n = 2. Thus, there is a factor of 10 decrease in the inactivation sensitivity of these repair-proficient strains when compared to the uvr- strain. Further, the values for n obtained are greater than 1.0, which implies that more than one hit is required. Inactivation could result from the lesion rendering ineffective, by whatever mechanism, any influence of an inducing protein. Inactivation could also result from a stabilized dimer within the prophage (15) interfering with transcription of essential genes or with replication of the prophage genome. Curves describing the UV inactivation of free phage particles, as assayed on uvr- and uvr+ hosts, were compared with those for inactivation of induced lysogens (Fig. 2). The slopes of the uvr- inactivation curves for free phage and lysogens differ by a factor of 1.33. We interpret these results to mean that the target size for rendering the lysogenic bacteria noninducible is approximately the same as the target size for inactivation of free phage particles. As we stated previously, the statistical approach of correlating expected frequencies of HPlcl induction with observed frequencies in a repair-deficient host as a function of UV dose leads us to consider that the occurrence of one dimer within a prophage-size (2.5 x 107 daltons) length of DNA triggers induction. Whether the 2.5 x 107-daltons length is that portion of the bacterial genome occupied by the prophage or is a segment occupied by nonphage
genes is, of course, conjecture. In 1954, Franklin (9) concluded that induction of phage X might involve the activation or inactivation of a particular enzyme-forming system. The appearance of an active inducing protein, as postulated by Tomizawa and Ogawa (17), could satisfy this possibility. Franklin (9) also offered the possibility that induction might result from the direct activation of provirus. Our results suggest that the induction response may indeed result from the appearance of UV photodimer(s) within the prophage genome of 2.5 x 107 daltons. This is the size segment of DNA that our results and correlations show to be "induction sensitive." Although the current dogma does not embrace the notion of prophage induction as being triggered by an initial event ocurring within the prophage genome, we feel that the close correlation of induction frequencies in a repair-deficient lysogen with dimer frequencies per prophage-size piece of the bacterial genome justifies a renewed consideration of induction being the result of a radiation insult primarily and directly to the prophage itself. We can only speculate as to the mechanism of action of the inducing lesion. One dimer within the prophage could render the repressoroperator complex (12) ineffective so that transcription and replication of phage genes proceed. Among the early proteins transcribed would be one that inactivates repressor protein in both the bound and unbound states. However, if induction is mediated by an inducing protein, as suggested by Tomizawa and Ogawa (17), the inducing dimer could permit a receptor on the prophage or on the repressor itself to become available to binding and thereby subject to the functional influence of the inducing protein. This protein could be produced as the result of UV lesions in any stretch of DNA, whereas a lesion specifically within the prophage would be necessary to permit such an inducing protein to function on the repressed operon. Whether induction involves recombinational repair, as described by Rupp and HowardFlanders (14), or an event similar to one of the steps in recombination that acts to lift phage immunity, is not known. Since H. influenzae is rec+ (15), it is possible that a gene product similar to that coded for by recA of E. coli acts on the UV lesion incurred by prophage HPlcl in a manner that permits prophage derepression. ACKNOWLEDGMENTS We thank Richard T. Okinaka of this laboratory and Thomas S. Matney of the M. D. Anderson Hospital and Tumor Institute, University of Texas, Houston, for helpful
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discussions while this work was in progress and during preparation of the manuscript. This work was performed under the auspices of the U.S. Energy Research and Development Administration. LITERATURE CITED 1. Alexander, H. E., and G. Leidy. 1953. Induction of streptomycin resistance in sensitive Henophilu inluenzae by extracts containing deoxyribonucleic acid
from resistant Henophilus influenzae. J. Exp. Med.
97:17-31. 2. Barnhart, B. J., and S. H. Cox. 1968. Radiation-sensitive
and radiation-resistant mutants of Haemophilus influenzae. J. Bacteriol. 96:280-282.
3. Barnhart, B. J., and S. H. Cox. 1970. Recovery of Haemophilus influenzae from ultraviolet and x-ray damage. Photochem. Photobiol. 11:147-162. 4. Brnhart, B. J., and S. H. Cox. 1970. Radiation sensitivity of Haemophilus influenzae: a composite response. J. Bacteriol. 103:9-15. 5. Barmhart, B. J., and R. M. Heriott. 1963. Penetration of
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deoyribonucleic acid into Haemophilus influenzae. Biochim. Biophys. Acta 76:25-39. Bern., H. I., and C. A. Thomas, Jr. 1965. Isolation of high molecular weight DNA from Haemophilus influenzae. J. Mol. Biol. 11:476490. Borek, E., and A. Ryan. 1960. The transfer of a biologically active irradiation product from cell to cell. Biochim. Biophys. Acta 41:67-73. Brooks, K., and A. J. Clark. 1967. Behavior of A bacteriophage in a recombination deficient strain of Esherichia coli. J. Virol. 1:283-293. Franklin, R. 1954. The action spectrum for the ultraviolet
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induction of lysis in Escherichia coli K-12. Biochim. Biophys. Acta 13:137-138. Gillis, M., J. DeLey, and M. DeCleeve. 1970. The determination of molecular weight of bacterial genome DNA from renaturation rates. Eur. J. Biochem. 12: 143-153. Hann, W., and C. S. Rupert. 1963. Infection of transormable cells of Haemophilus influerzae by bacteriophage and bacteriophage DNA. Z. Vererbungpl. 94:336-348. Jacob, F., and J. Monod. 1961. Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3:318-356. Mattern, I. E., M. P. Van Winden, and A. Rorwch. 1965. The range of action of genes controlling radiation sensitivity inEscherichia coli. Mutat. Res. 2:111-131. Rup, D., and P. Howard-Flanders. 1968. Disa6ntinuities in the DNA synthesized in an excision-defective strain of Escherichia col following ultraviolet irradiation. J. Mol. Biol. 31:291-304. Setlow, J. K., M. E. Boling, D. P. Allison, and K. L. Beattie. 1973. Relationship between prophage induction and transfornation in Haemophilus influenzae. J. Bacteriol. 115&153-161. Setlow, R. B., P. A. Swenson, and W. L. Carrier. 1963. Thymine dimers and inhibition of DNA synthesis by ultraviolet irradiation of cells. Science 142:1464-1466. Tonizawa, J. I., and T. Ogawa. 1967. Effect of ultraviolet irradiation on bacteriophage lamnbda immunity. J. Mol. Biol. 23:247-263. Wulff, D. L. 1963. The role of thymine dimer in the photo-inactivation of the bacteriophage T4V,. J. Mol. Biol. 7:431-441.
